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Hybrid three-dimensional (3D) bioprinting of retina equivalent for ocular research

2.5 Y79 cell bioprinting 3. Results
Y79 cells were in a suspension culture, and the cells The whole bioprinting strategies for retinal regeneration
were counted before collection. The cell pellet was re- could be clearly observed in Figure 1. The bioprinting
suspended in alginate/pluronic bioink to obtain a final protocol was designed to precisely and efficiently
6
cell density of 1 × 10 cell/ml. The Y79 cell-laden bioink simulate the biological functions of native retina. The
was filled in a cartridge and kept at 37 °C. The cartridge ARPE-19 cells were precisely bioprinted on the ultrathin
was connected to a 21G needle tip, and pressure (2 bars) membrane at discrete places to obtain homogenous cell
was applied to motivate the bioink to go through the seeding, and then the cells were allowed to grow for two
needle to print two distinctive patterns on the ARPE- weeks until the formation of ARPE-19 cell monolayer.
19 cell-seeded ultrathin membrane: a high average The Y79 cell-laden alginate/pluronic bioink were
cell density at the center (HC) and a high average cell bioprinted on the ARPE-19 cell monolayer to achieve
density at the periphery (HP). The bioprinted Y79 cell- two different cell-seeding densities, as can be seen in
laden bioink was crosslinked in 50 mM calcium chloride Figure 1 c1 and c2. The bioprinted ARPE-19 cells were
solution for 5 mins. Subsequently, the retina equivalents closely monitored at day 1, 7 and 14 via prestoblue
were cultured in cell culture media. assay (Figure 2). The trend of cell viability in the
bioprinted samples were similar to that of the control,
2.6 Bioprinted retina equivalent characterization their values increased gradually from day 1 to day 14,

The cell viabilities of bioprinted ARPE-19 cells at day and no significant differences were observed in both
1, 7 and 14 were evaluated by prestoblue (Thermo groups. In Figure 3, the ARPE-19 cell morphology and
Fisher, Grand Island, NY, USA) assay in test media proliferation were investigated, and the cells attached to
(1 ml) consisted of prestoblue (10%) and FBS (5%). the membrane within 24 hours, and then migrated and
Control groups were cells without bioprinting. Then, spread on the membrane. The cell number increased
the cell viabilities were calculated according to vendor’s markedly, and finally the bioprinted ARPE-19 cells
protocol. Meanwhile the bioprinted ARPE-19 cells at formed cell monolayer at day 14. The bioprinted ARPE-
day 1, 7 and 14 were fixed in 4% paraformaldehyde; 19 cells on the ultrathin membrane at day 14 were
then, they were stained by ActinGreen™ 488 further analyzed by inverted microscope, and the bright
ReadyProbes® and NucBlue® Live ReadyProbes® field image and HE staining indicated that the bioprinted
cells formed an intact cell monolayer on the ultrathin
reagents and observed under inverted microscope membrane (Figure 4). The bioprinted ARPE-19 cells on
(Zeiss). The bioprinted ARPE-19 cells on ultrathin ultrathin membrane were investigated under confocal
membrane at day 14 were also observed under inverted microscope (Figure 5). It can be observed that the strong
microscope, and the sample was then fixed by 4% actin filaments were within each cell, and the cells were
paraformaldehyde and HE stained. The bioprinted closely packed with polygonal appearance. Moreover,
sam ples were stained by ZO-1 Monoclonal Antibody, no cell clusters and aggregations were observed on
FITC (ZO1-1A12)/NucBlue® Live ReadyProbes® the ultrathin membrane at week 2. Furthermore, the
reagents and ActinGreen™ 488 ReadyProbes®/ bioprinted ARPE-19 cells were stained by ZO-1
NucBlue® Live ReadyProbes® reagents and live/dead antibodies (Figure 6), and it showed that robust tight
assay, respectively. The stained samples were observed junctions existed within the ARPE19 cell monolayer.
under inverted microscope (Zeiss) and laser scanning The Y79 cell-laden alginate/pluronic bioink was
microscope (Zeiss LSM 710). For scanning electron printed on the ultrathin membrane upon the formation
microscope (SEM, JEOL) observation, the samples of ARPE-19 cell monolayer. In Figure 7, the cell-laden
were fixed at day 1, 7 and 14, and then were dehydrated bioink was bioprinted into two distinctive patterns: the
in 30%, 50%, 75%, 95% and 100% ethanol gradually first one is with the higher average Y79 cell-seeding
before critical-point drying, and the samples were density at the center, and the other one is the higher
sputter-coated (sputtering time 90 s and current 20 mA) average Y79 cell-seeding density at the periphery. The
by gold before SEM observation. samples in both groups were maintained in culture media
2.7 Statistical analysis for seven days. The Y79 cells in alginate/pluronic bioink
after bioprinting were evaluated by live/dead assay, and
All data was presented as means ± standard deviation the live cells were stained in green while dead cells were
(n = 3). Statistical analysis was implemented by paired in red color (Figure 8). Most of the Y79 cells survived
samples t-test and multiple comparisons using single- at day 1, and they proliferated obviously at day 4 and 7
factor analysis of variance (ANOVA) and post-hoc with high cell viability. Subsequently, the bioprinted Y79
Tukey tests using SPSS Statistics version 19.0, and p < cell-laden constructs were observed under SEM (Figure
0.05 was considered statistically significant. 9): the width of each construct was around 0.21 mm, and

140 International Journal of Bioprinting (2017)–Volume 3, Issue 2